Reconstruction of urologic structures with polymeric matrices

ABSTRACT

A method for repairing defects and reconstructing urothelial structures in vivo has been developed using a fibrous, open synthetic, biodegradable polymeric matrix which is configured to provide the desired corrective structure. The matrix is shaped to correct the defect, then implanted surgically to form a scaffolding for the patient&#39;s own cells to grow onto and into. The implantation of the matrix initiates an inflammatory reaction, resulting in urothelial cells, endothelial cells and mesenchymal cells, to migrate into the matrix. The polymer forming the matrix is selected to be biocompatible and degradable in a controlled manner over a period of one to six months, in the preferred embodiment. A preferred material is a poly(lactic acid-glycolic acid) in a fibrous form, such as a woven or non-woven mesh. Examples demonstrate the repair of defects in bladder in rabbits.

BACKGROUND OF THE INVENTION

[0001] The present invention is generally in the area of methods for reconstruction of urothelial structures, especially bladders.

[0002] Traditionally, defects in the bladder and other urothelial structures have been corrected surgically. This has obvious disadvantages when there is a defect in the structure which requires closure of an opening for which there is insufficient tissue or when the structure itself is deformed or too small to meet the needs of the patient.

[0003] Bowel segments have been used in reconstruction of genitourinary structures in these circumstances. The use of bowel in genitourinary reconstruction is associated with a variety of complications, including metabolic abnormalities, infection, perforation, urolithiasis, increased mucus production and malignancy, as reviewed by Atala, A. and Retik, A.: Pediatric urology—future perspectives. In: Clinical Urology. Edited by R/J. Krane, M. B. Siroky and J. M. Fitzpatrick. (Philadelphia: J.B. Lippincott, 1993). Alternative approaches need to be developed to overcome the problems associated with the incorporation of intestinal segments into the urinary tract. Natural tissues and synthetic materials that have been tried previously in experimental and clinical settings include omentum, peritoneum, seromuscular grafts, de-epithelialized segments of bowel, polyvinyl sponge and polytetrafluoroethylene (Teflon). These attempts have usually failed.

[0004] It is evident that urothelial-to-urothelial anastomoses are preferable functionally. However, the limited amount of autologous urothelial tissue for reconstruction generally precludes this option. In cell transplantation, donor tissue is dissociated into individual cells or small tissue fragments and either implanted directly into the autologous host or attached to a support matrix, expanded in culture and reimplanted after expansion. Autologous skin cells have been used in this fashion in the treatment of extensive burn wounds, as reported by Green, et al., “Growth of cultured human epidermal cells into multiple epithelia suitable for grafting”, Proc. Natl. Acad. Sci., 76:5665 (1979); O'Connor, et al., “Grafting of burns with culture epithelium prepared from autologous epidermal cells”, Lancet, 1:75 (1981); and Burke, et al., “Successful use of a physiologically acceptable artificial skin in the treatment of an extensive burn injury”, Ann. Surg., 194:413 (1981).

[0005] A suitable material for a cell transplantation matrix must be biocompatible to preclude migration and immunological complications, and should be able to support extensive cell growth and differentiated cell function. It must also be resorbable, allowing for a completely natural tissue replacement. The matrix should be configurable into a variety of shapes and should have sufficient strength to prevent collapse upon implantation. Recent studies indicate that the Biodegradable polyester polymers made of polyglycolic acid seem to fulfill all of these criteria, as described by Vacanti, et al., “Selective cell transplantation using bioabsorbable artificial polymers as matrices”, J. Ped. Surg., 23:3 (1988); Cima, et al., “Hepatocyte culture on biodegradable polymeric substrates”, Biotechnol. Bioeng., 38:145 (1991); Vacanti, et al., “Synthetic polymers seeded with chondrocytes provide a template for new cartilage formation”, J. Plast. Reconstr. Surg., 88:753 (1991).

[0006] The feasibility of using biodegradable polymers as delivery vehicles for urothelial cell transplantation has been demonstrated by studies showing that urothelial cells will adhere to synthetic polymers composed of polyglycolic acid and survive in vivo, as reported by Atala, et al., “Formation of urothelial structures in vivo from dissociated cells attached to biodegradable polymer scaffolds in vivo”, J. Urol., part 1, 148:658 (1992).

[0007] For implantation of cells on polymer matrices to be successful in patients, a source of an effective concentration of cells has to be available, and the urothelial cell population has to survive for extended times on implanted polymers and proliferate extensively in vivo. Most importantly, implanted cells have to remain intact as defined structures as the polymer implant degrades over time under physiological conditions. Polymer scaffolds would have to include bladder smooth muscle in concert with urothelial cells to reconstitute a functional bladder wall.

[0008] An easier solution would be to develop a method for correcting defects which did not require obtaining and implanting cells on the polymer matrices. However, initial studies with chondrocytes implanted in tissue in the absence of a matrix and implantation of polymer alone has not been demonstrated to result in appropriate ingrowth and proliferation of cells.

[0009] It is therefore an object of the present invention to provide a method and means for reconstructing defects in organ structures, especially urothelial structures such as the bladder, ureter and urethra, which does not require exogenous cells.

SUMMARY OF THE INVENTION

[0010] A method for repairing defects and reconstructing urological structures in vivo has been developed using a fibrous, open, synthetic, biodegradable polymeric matrix. The matrix is shaped to correct the defect, then implanted surgically to form a scaffolding for the patients own cells to grow onto and into. The implantation of the matrix initiates an inflammatory reaction, resulting in urothelial cells, including both endothelial cells and mesenchymal cells, migrating into the matrix. The polymer forming the matrix is selected to be biocompatible and degradable in a controlled manner over a period of one to six months in the preferred embodiment. A preferred material is a polyhydroxy acid, poly(lactic acid-glycolic acid), in a fibrous form, such as a woven or non-woven mesh.

[0011] Examples demonstrate the repair of defects in bladders in rabbits.

DETAILED DESCRIPTION OF THE INVENTION

[0012] Previous studies have indicated that cells implanted in the absence of a matrix and that matrices implanted in the absence of seeded cells do not form structures. In contrast, previous studies have indicated that very small repairs can be achieved by covering the defect with a “patch” or other biodegradable or non-degradable mesh, so that the surrounding tissue grows over the defect. The usefulness of polymeric matrices, in the absence of seeded cells, either before or after implantation of the matrix, to form tissue structures, is surprising. Based on the previous studies, one would have expected problems, including compression of the matrix after surgical attachment which would prevent cells from entering into and proliferating in the matrix to form tissue; migration into and proliferation within the matrix of the wrong cell populations; and/or that the matrix would have detached or degraded prior to tissue formation. As demonstrated by the following examples, none of these problems occurred and the materials did form tissue that effectively repaired the defects in bladders.

[0013] Polymeric Materials

[0014] A variety of polymeric materials can be used to make the matrix. In the preferred embodiment, the material is biocompatible, biodegradable over a period of one to six months, synthetic, and easily fabricated. The most preferred material is poly(lactic acid-glycolic acid).

[0015] In the preferred embodiment, the matrix is formed of a bioabsorbable, or biodegradable, synthetic polymer such as a polyanhydride, polyorthoester, polyhydroxy acid, for example, polylactic acid, polyglycolic acid, and copolymers or blends thereof, and polyphosphazenes. Collagen can also be used, but is not as controllable as a synthetic polymer either with respect to manufacture of matrices or degradation in vivo and is therefore not preferred. These materials are all commercially available.

[0016] In some embodiments, attachment of the cells to the polymer is enhanced by coating the polymers with compounds such as basement membrane components, agar, agarose, gelatin, gum arabic, collagens types I, II, III, IV and V, fibronectin, laminin, glycosaminoglycans, mixtures thereof, and other materials known to those skilled in the art of cell culture.

[0017] All polymers for use in the matrix must meet the mechanical and biochemical parameters necessary to provide adequate support for the cells with subsequent growth and proliferation. The polymers can be characterized with respect to mechanical properties such as tensile strength using an Instron tester, for polymer molecular weight by gel permeation chromatography (GPC), glass transition temperature by differential scanning calorimetry (DSC) and bond structure by infrared (IR) spectroscopy, with respect to toxicology by initial screening tests involving Ames assays and in vitro teratogenicity assays, and implantation studies in animals for immunogenicity, inflammation, release and degradation studies.

[0018] One of the advantages of a biodegradable polymeric matrix is that angiogenic and other bioactive compounds can be incorporated directly into the matrix so that they are slowly released as the matrix degrades in vivo. As the cell-polymer structure is vascularized and the structure degrades, the cells will differentiate according to their inherent characteristics. Factors, including nutrients, growth factors, inducers of differentiation or de-differentiation (i.e., causing differentiated cells to lose characteristics of differentiation and acquire characteristics such as proliferation and more general function), products of secretion, immunomodulators, inhibitors of inflammation, regression factors, biologically active compounds which enhance or allow ingrowth of the lymphatic network or nerve fibers, hyaluronic acid, and drugs, which are known to those skilled in the art and commercially available with instructions as to what constitutes an effective amount, from suppliers such as Collaborative Research, Sigma Chemical Co., vascular growth factors such as vascular endothelial growth factor (VEGF), EGF, and HB-EGF, could be incorporated into the matrix or provided in conjunction with the matrix. Similarly, polymers containing peptides such as the attachment peptide RGD (Arg-Gly-Asp) can be synthesized for use in forming matrices.

[0019] A presently preferred polymer is polyglactin 910, developed as absorbable synthetic suture material, a 90:10 copolymer of glycolide and lactide, manufactured as Vicryl® braided absorbable suture (Ethicon, Inc., Somerville, N.J.) (Craig, P. H., Williams, J. A., Davis K. W., et al.: A Biological Comparison of Polyglactin 910 and Polyglycolic Acid Synthetic Absorbable Sutures. Surg., 141:1010 (1975). A commercially available surgical mesh formed of polyglycolic acid, Dexon™, can also be used.

[0020] Matrix Design

[0021] The design and construction of the scaffolding is of primary importance.. The matrix should be a pliable, non-toxic, injectable porous template for vascular ingrowth. The pores should allow vascular ingrowth. These are generally interconnected pores in the range of between approximately 100 and 300 microns, i.e., having an interstitial spacing between 100 and 300 microns, although larger openings can be used. The matrix should be shaped to maximize surface area, to allow adequate diffusion of nutrients, gases and growth factors to the cells on the interior of the matrix and to allow the ingrowth of new blood vessels and connective tissue. At the present time, a porous structure that is relatively resistant to compression is preferred, although it has been demonstrated that even if one or two of the typically six sides of the matrix are compressed, that the matrix is still effective to yield tissue growth.

[0022] Fibers (sutures or non-woven meshes) can be used as supplied by the manufacturer. Other shapes can be fabricated using one of the following methods:

[0023] Solvent Casting. A solution of polymer in an appropriate solvent, such as methylene chloride, is cast on a fibrous pattern relief structure. After solvent evaporation, a thin film is obtained.

[0024] Compression Molding. Polymer is pressed (30,000 psi) into an appropriate pattern.

[0025] Filament Drawing. Filaments are drawn from the molten polymer.

[0026] Meshing. A mesh is formed by compressing fibers into a felt-like material.

[0027] At the present time, a mesh-like structure formed of fibers which may be round, scalloped, flattened, star shaped, solitary or entwined with other fibers is preferred. As discussed above, the polymeric matrix may be made flexible or rigid, depending on the desired final form, structure and function. either woven, non-woven or knitted material can be used. A material such as a velour is an example of a suitable woven material. The fibers can be fused together by addition of a solvent or melting to form a more stable structure. Alternatively, high pressure jets of water onto a fibrous mat can be used to entangle the fibers to form a more rigid structure. For repair of a defect, for example, a flexible fibrous mat is cut to approximate the entire defect, then fitted to the surgically prepared defect as necessary during implantation. An advantage of using the fibrous matrices is the ease in reshaping and rearranging the structures at the time of implantation.

[0028] A sponge-like structure can also be used. The structure should be an open cell sponge, one containing voids interconnected with the surface of the structure, to allow adequate surfaces of attachment for sufficient cells to form a viable, functional implant.

[0029] Implantation of the Matrix

[0030] The matrix is implanted using standard surgical procedures, suturing edges to the tissue to be treated or adjacent materials as necessary.

[0031] This method of using a polymer as a scaffold wherein adjacent cells can migrate onto and into the polymer can be used to patch defects of urethelial associated organs such as urethra, bladder, ureters, and renal pelvis. In addition, this method can be used to entirely replace or reconstruct these structures, such as for hypospadias, where urethral reconstructive surgery is necessary, or for bladder surgery where either an augmentation is necessary for a low capacity bladder or a neobladder is needed, or for ureteral extension, replacement, or reconstruction, such as with a patient requiring additional ureteral length secondary to trauma or neoplasm. Further, this system can be used for other areas where a soft tissue replacement is needed such as in the gastrointestinal system, for example, in situations where additional intestinal tissue is needed, or in the musculo-skeletal system, such as for bone or cartilage tissue replacement secondary to congenital, neoplastic, inflammatory, or traumatic conditions.

[0032] The present invention will be further understood by reference to the following non-limiting examples.

EXAMPLE 1

[0033] Urethral Reconstruction Using Biodegradable Polymer Scaffolds.

[0034] The more severe forms of hypospadias are usually corrected with a vascularized preputial island graft. Patients with failed reconstruction, epispadias, or urethral strictures may not have sufficient preputial skin for repair. In these instances, several alternatives have been used, including free skin, bladder mucosa and buccal mucosa grafts. However, some of these grafts are associated with several complications, and their use is limited. The following study compares the usefulness of cell-polymer matrices and synthetic polymer matrices in the absence of seeded cells for repair of urothelial structures, especially bladder.

[0035] Materials and Methods

[0036] Urothelial cells were harvested from a small segment of the bladders of 10 New Zealand white rabbits by open surgery. The urothelial cells were plated in vitro, expanded, and tagged with 7-amino 4-chloromethylcoumarin, a fluorescent probe. Cells were resuspended in media and seeded onto biodegradable polymer scaffolds. Partial urethrectomies were performed in each rabbit through a circumcising incision. The autologous urothelial cell-polymer meshes were interposed using continuous 7-0 Vycril™ sutures to form the neourethras. Polymer meshes without urothelial cells were used in two animal as controls. The penile skin was closed over the neourethra with interrupted 5-0 Vycril™ sutures. Due to the thick, semi-solid consistency of rabbit urine, simultaneous vesicotomies were performed in order to achieve a satisfactory urinary diversion.

[0037] After vesicotomy closure, ten days after urethral reconstruction, the animals were able to void through the neourethra without complications. Retrograde urethrograms showed no evidence of stricture formation. Histologic examination of the neourethras demonstrated complete re-epithelialization of the polymer mesh sites by day 14. These findings were persistent at the four and six week time points. Fluorescent microscopy showed tagged autologous urothelial cells closely associated with the poly fibers. Urethral polymer controls showed complete re-epithelialization, by 14 days, indicating that native cells are not necessary for successful replacement of urethral defects. The polymer fibers were partially degraded by day 14 and almost totally reabsorbed by day 30.

[0038] In conclusion, biodegradable polymer meshes can be used, either alone or in combination with harvested autologous urothelial cells, for urethral reconstruction. Adequate anatomic and functional replacement can be achieved by using this technology.

EXAMPLE 2

[0039] Bladder Reconstruction Using Biodegradable Polymer.

[0040] Multiple anomalies of the bladder, whether congenital or acquired, require augmentation cystoplasty. In these instances, use of bowel for augmentation has been used widely. The use of gastrointestinal tissue for urologic reconstruction is associated with several complications, and their use is limited. The following study compares the usefulness of cell polymer matrices and cell-polymer matrices and synthetic polymer matrices in the absence of seeded cells for bladder reconstruction.

[0041] Urothelial cells are harvested from a small segment of the bladders of ten New Zealand white rabbits by open surgery. The urothelial cells were plated in vitro and tagged with 7-Amino 4-chloromethylcomarin, a fluorescent probe. Cells were resuspended in media and seeded onto biodegradable polymer scaffolds. Partial cystectomies were performed in each rabbit through a mid-abdominal incision. The autologous urothelial cell-polymer matrices were interposed using Vycril™ sutures to augment the small bladders. Polymer matrices without urothelial cells were used in ten additional animals as controls. Omentum was used to cover the polymer, rendering it impermeable to urine. A urethral catheter was left in place for ten days for urinary diversion.

[0042] After the catheter was removed, the animals were able to void without complications. Bladder cystograms showed an increased bladder capacity in all animals. Histological examination of the neobladders demonstrated complete re-epithelialization of the polymer mesh sites by day 14. These findings were persistent at the four and six week time points. Fluorescent microscopy showed tagged autologous urothelial cells closely associated with the polymer fibers. Bladder polymer controls showed complete re-epithelialization by 14 days, indicating that native cells are not necessary for successful bladder augmentation or reconstruction. The polymer fibers were partially degraded by day 14 and almost totally reabsorbed by day 30.

[0043] In conclusion, biodegradable polymer matrices can be used, either alone or in combination with harvested autologous urothelial cells, for bladder reconstruction. Adequate anatomic and functional replacement can be achieved by using this technology.

EXAMPLE 3

[0044] Ureteral Reconstruction Using Biodegradable Polymers Scaffolds.

[0045] Multiple anomalies of the ureter, whether congenital or acquired, require ureteral reconstruction. In these instances, when the ureteral tissue present cannot be used for reconstruction, other gastrointestinal tissues have been used. The use of gastrointestinal tissue, however, is associated with numerous complications when they are interposed with the urinary tract. The following study compares the usefulness of cell polymer matrices and synthetic polymer matrices in the absence of seeded cells for bladder reconstruction.

[0046] Urothelial cells were harvested from a small segment of the bladder of ten beagle dogs by open surgery. The urothelial cells were plated in vitro, expanded, and tagged with 7-amino 4-chloromethylcomarin, fluorescent probe. Cells were resuspended in media and seeded onto biodegradable polymer scaffolds. Partial ureterectomies were performed in each dog for a flank incision. The autologous urothelial cell-polymer matrices were interposed using Vicryl™ sutures to interpose these ureters. Polymer matrices without urothelial cells were used in 10 additional animals as controls. Gerota's fascia was used to cover the polymer, rendering it impermeable to urine. A ureteral catheter was left in place indwelling for ten days for urinary diversion.

[0047] After the catheter was removed, an intravenous pyelogram was performed which showed normal ureteral anatomy in each animal, without any evidence of obstruction. Histological examination of the neo-ureters demonstrated complete epithelialization of the polymer mesh sites by day 14. These findings were persistent at the 4th and 6th week time points. Fluorescent microscopy showed tagged autologous urethelial cells closely associated with the polymer fibers. Bladder polymer control showed complete re-epithelialization by 14 days, indicating that native cells are not necessary for successful ureteral reconstruction. The polymer fibers were partially degraded by day 14 and almost totally reabsorbed by day 30.

[0048] In conclusion, biodegradable polymer matrices can be used, either alone or in combination with harvested autologous urethelial cells, for ureteral reconstruction in large animals.

[0049] Although this invention has been described with reference to specific embodiments, variations and modifications of the method and means for constructing urothelial implants by implantation of polymeric matrices will be apparent to those skilled in the art. Such modifications and variations are intended to come within the scope of the appended claims. 

We claim:
 1. A method for correcting urothelial defects comprising implanting at the site to be corrected a matrix of a biodegradable, biocompatible synthetic polymeric fibers forming a matrix having an interstitial spacing of at least 100 microns, shaped to correct the defect.
 2. The method of claim 1 wherein the defect is in the bladder.
 3. The method of claim 1 wherein the defect is in the ureter or urethra.
 4. The method of claim 1 wherein the defect is in the gastrointestinal tract.
 5. The method of claim 1 further comprising providing with the matrix factors selected from the group consisting of nutrients, growth factors, inducers of differentiation or de-differentiation, products of secretion, immunomodulators, inhibitors of inflammation, biologically active compounds which enhance or allow ingrowth of the lymphatic network or nerve fibers, vascular growth factors, attachment peptides, and combinations thereof.
 6. The method of claim 1 wherein the matrix is coated with a material selected from the group consisting of basement membrane components, agar, agarose, gelatin, gum arabic, collagens types I, II, III, IV and V, fibronectin, laminin, glycosaminoglycans, and mixtures thereof.
 7. A fibrous matrix formed of biodegradable synthetic polymer having an interstitial spacing between 100 and 300 microns which is shaped to repair a tissue defect.
 8. The matrix of claim 7 wherein the defect is in a urological structure.
 9. The matrix of claim 8 where the urological structure is selected from the group consisting of ureters, urethras, bladders and renal pelvis.
 10. The matrix of claim 7 wherein synthetic polymer is selected from the group consisting of polyanhydrides, polyorthoesters, polyphosphazenes, and polyhydroxy acids.
 11. The matrix of claim 10 wherein the polymer is a polyhydroxy acid is selected from the group consisting of polylactic acid, polyglycolic acid, and copolymers thereof.
 12. The matrix of claim 7 wherein the matrix degrades over a period of between one and six months following implantation. 